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Cure Arthritis Naturally

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Amplified fragment-length polymorphism analysis (AFLP) has been shown to be a suitable method for subtyping of bacteria belonging to the genus Campylobacter. Campylobacter lari is a phenotypically and genotypically diverse species that comprises the classical nalidixic acid-resistant thermophilic campylobacters and the biochemical C. lari variants, urease-positive, nalidixic acid-susceptible, and urease producing nalidixic acid-susceptible strains. AFLP profiling and whole-cell protein profile analysis are suitable methods for studying the taxonomic and epidemiological relationships among strains of the C. lari variants. Numerical analysis of AFLP profiles and of partial protein profiles allows the discrimination of distinct C. lari genogroups. No correlation of these genogroups with different sources of the strains has been identified until now.

Key Words: Campylobacter lari; AFLP typing; genogroups; protein profiling. 1. Introduction

Campylobacter species are important pathogens that cause a variety of diseases in humans and animals (1,2). The most frequent found are the thermophilic species Campylobacter jejuni, C. coli, C. upsaliensis, and C. lari (3). C. lari has been associated in humans with diarrhoea (3), bacteraemia in immunocompromised and immunocompetent patients (4,5), urinary tract infection (6), reactive arthritis (7) and, recently, with a prosthetic joint infection (8). Infections after consumption of contaminated shellfish as well as a large outbreak from a waterborne common source have been reported (9,10). Outbreaks caused by C. lari incidentally are reported and it is assumed that the number of C. lari infections is

FFrom: Methods in Molecular Biology, vol. 345: Diagnostic Bacteriology Protocols, Second Edition Edited by: L. O'Connor © Humana Press Inc., Totowa, NJ

highly underreported as the isolation methods for Campylobacter species are developed for the detection of C. jejuni and C. coli (11). C. lari is widely distributed in the environment and can be isolated from a variety of sources including water and animals. Seagulls have been shown to be a reservoir for C. lari and have been proposed to contribute to the contamination of water storage reservoirs and of mussels and oyster banks (12,13).

Since the late 1980s, the identification of C. lari has been performed using nalidixic acid with the production of urease. Primarily nalidixic acid-resistant thermophilic Campylobacter (NARTC group) and nalidixic acid-susceptible strains (NASC strains) were identified. Subsequently, urease-producing strains (UPTC strains) and urease-producing nalidixic acid-susceptible strains (UP-NASC [13-15]) were identified.

Because the phenotypic diversity and the contribution of C. lariphenotypes in human infection is incompletely understood, characterization of the C. lari strains that are present in the environment and in human diseases is important. Furthermore, typing may identify strain or group specific markers which may contribute to their pathogenic potential. We describe in this chapter the method we have developed for molecular identification and typing of C. lari isolates obtained from various sources (birds, environmental samples, and human patients). Once bacterial strains are isolated they are typed with amplified fragment-length polymorphism analysis (AFLP) and protein profile analysis. The AFLP technique relies on selective amplification of restriction fragments from a digest of genomic deoxyribonucleic acid (DNA). Two restriction enzymes are used to digest DNA, giving rise to fragments of a size (often 50-500 bp) suitable for resolution on polyacrylamide gel or capillary DNA sequencers. Double-stranded adapters, specific for either restriction site are ligated to the DNA fragments, serving as target sites for primers used in PCR amplification. To reduce the number of amplified bands, selective nucleotides can be synthesized at the 3' ends ofthe PCR primers, allowing amplification of only a subset of the DNA molecules (see Fig. 1). Initially, the AFLP utilized radioactively labeled primers for PCR amplification of small genomic fragments (16). More recently, fluorescent-labeled AFLP patterns are analyzed on gel-based or capillary DNA sequencers, with the corresponding advantage being that the resulting fingerprints may be directly and easily imported into software programs and stored in databases for further processing, for example, comparisons of similarity.

We have adapted the AFLP method for typing of Campylobacter strains, and identification of Campylobacter species (17). The AFLP method appeared to correlate with typing date obtained with other genetic typing methods (18) and was demonstrated to consist of a good discriminatory power for typing of C. lari strains (19).

Amplified Fragment Length Polymorphism

Fig. 1. Schematic representation of the amplified fragment-length polymorphism method. In the adapter sequence ('), a point mutation (in bold) is incorporated to prevent digestion after ligation. Both polymerase chain reaction primers contain one selective nucleotide (in bold). One primer is fluorescently labeled (*). (From ref. 19 with permission from ASM.)

Fig. 1. Schematic representation of the amplified fragment-length polymorphism method. In the adapter sequence ('), a point mutation (in bold) is incorporated to prevent digestion after ligation. Both polymerase chain reaction primers contain one selective nucleotide (in bold). One primer is fluorescently labeled (*). (From ref. 19 with permission from ASM.)

Polyacrylamide gel electrophoresis (PAGE) in the presence of the detergent sodium dodecylsulfate (SDS) has proved to be an important method for the analysis of the protein composition of tissues, cells or biological membranes. From the mid 1970s, electrophoresis of whole-cell lysate fractions in slab gels became an attractive tool for the taxonomic positioning and epidemiological typing of microorganisms and standardized protocols arose to make gels amenable for computerized analysis (20,21). Currently, SDS-PAGE still holds an important place in the taxonomic characterization of microorganisms to strain level and species (22). In this chapter, we describe a method for protein profile analysis of C. lari. Analysis of a protein range with numerical analysis enables the identification of C. lari genogroups (19).

AFLP has the advantage that it samples a random proportion of the whole genome and therefore has the potential to provide high-resolution epidemio-logical typing. With AFLP a genotype of an individual C. lari is obtained that may cluster into different genotypes. The method described in this chapter is suitable for typing of strains of several Campylobacter species. In general, the AFLP method is applicable to almost all microorganisms without previous knowledge of chromosomal DNA or restriction enzyme digestion sites. Moreover, the method is straightforward and can be completed within 8 to 48 h (depending on the electrophoresis platform used). The most important control steps in the method are the purity and quantity of DNA. Data analysis may be performed using software packages as patterns from polyacrylamide gels or capillary sequencers are too complicated to analyse manually. Analysis of protein profiles, with numerical analysis of restricted parts of the profiles results in clustering of C. lari strains (see Fig. 2). When a similar clustering of strains is obtained by phenotypic and genotypic methods, it supports the definition of a specific genogroup. In summary, AFLP and protein analysis are both methods that are general and reproducible, which can be performed by any laboratory.

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